Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infections (PJIs) typified by an anti-inflammatory cellular milieu. The inflammatory phenotype of leukocytes is intimately tied to their metabolic status, where anti-inflammatory M?s primarily rely on oxidative phosphorylation (OxPhos) and pro-inflammatory M?s utilize glycolysis. Monocytes are also polarized toward an anti-inflammatory state during S. aureus PJI, which our preliminary data shows coincides with an OxPhos bias. We have devised an innovative approach to metabolically re-reprogram biofilm-associated monocytes to promote glycolysis using cell-targeted nanoparticles containing the OxPhos inhibitor oligomycin. Treatment of established biofilms with oligomycin nanoparticles promoted monocyte pro-inflammatory activity concomitant with increased neutrophil recruitment, leading to biofilm clearance. During the recent PPG cycle, our laboratory was the first to identify that myeloid- derived suppressor cells (MDSCs) skew biofilm-associated monocytes toward an anti-inflammatory state, in part, through IL-10 production. Therefore, we screened the Nebraska Transposon Mutant Library to identify mutants impaired in their ability to trigger IL-10 production. Significant hits involved in lactate biosynthesis were identified, and our preliminary data support a role for S. aureus-derived lactate in organizing the anti-inflammatory biofilm milieu, progressing from MDSCs/monocytes as a target to defining the molecular mechanism of action. First, during PJI, D- and L-lactate levels are reduced in S. aureus ddh1 and ldh1/ldh2 mutants, respectively, concomitant with significant reductions in MDSC infiltrates and IL-10 production, which translates into enhanced leukocyte recruitment, and biofilm clearance. Second, the IL-10 promoter is activated by acetylation and our ChIP-Seq data demonstrate that histone promoter acetylation is dramatically increased genome-wide in leukocytes recovered from WT vs. ldh1/ldh2 infected mice, providing molecular evidence that S. aureus biofilm- derived lactate functions as a histone deacetylase inhibitor (HDACi). Our central P01 hypothesis is that S. aureus biofilm development creates unique metabolic niches that promote an immune suppressive environment. In Project 4, we will explore the existence of a metabolic triad between S. aureus biofilm, MDSCs, and monocytes, whereby biofilm-derived lactate promotes leukocyte anti-inflammatory properties, in part, via IL- 10 production, contributing to biofilm persistence. This metabolic crosstalk and the molecular mechanisms responsible for this interplay will be explored in the following Specific Aims. 1) Establish that leukocyte metabolism can be targeted in vivo to promote pro-inflammatory activity and biofilm clearance; 2) Investigate the role of S. aureus biofilm-derived lactate in promoting MDSC and monocyte anti-inflammatory activity by stimulating IL-10 production; and 3) Determine whether S. aureus biofilm-derived lactate regulates IL-10 production by inhibiting histone deacetylase (HDAC) activity. These studies will inform our long-term goal of targeting metabolic pathways that disarm anti-bacterial innate immune defenses to facilitate biofilm eradication.